Recovery method for immobilized biocatalysts

ABSTRACT

Processes for the production of a product by the enzymatic treatment of a soluble or particulate substrate with a particulate, immobilized enzyme, by treating a process liquor containing the substrate in a bioreactor to produce a slurry of effluent immobilized enzyme and the product in an effluent liquor. The slurry is subject to a non-immobilized enzyme damaging shear inducing effective separation process to provide effluent immobilized enzyme, and effluent liquor containing the product; and reusing the effluent immobilized enzyme in the enzymatic treatment. The process provides for the reclamation and reuse of the immobilized enzyme even when a further particulate solid is present in the effluent/product stream.

RELATED APPLICATION

This U.S. Application claims the foreign priority under 35 U.S.C. §119,including subsections (a)–(d) and (f), from Canadian Application2,354,782, filed Aug. 2, 2001.

FIELD OF THE INVENTION

This invention relates to the use of immobilized enzymes on a matrix inchemical processes, particularly industrial processes, and moreparticularly to the recovery of said immobilized enzymes from saidprocess for reuse.

BACKGROUND OF THE INVENTION

The industrial use of enzymes is often limited by their high cost andrapid inactivation. Soluble enzymes are lost with the product at theconclusion of a process, and must be replenished.

One means to improve the economic feasibility of enzymes for industrialprocesses is through enzyme immobilization onto a matrix, which mayfacilitate their re-use. Immobilization research has focused upon meansto enhance the transfer of enzymes onto the support, and upon means toensure that the immobilized enzymes remain active. Inactivation ofenzymes during catalytic turnover is another key obstacle which limitsthe economic feasibility of enzyme-mediated processes. Enzymes may beinactivated by extremes of temperature, pH, shear, and also by freeradicals and other reactive species present in the reaction medium.Immobilization technology has the potential to reduce such enzymeinactivation, and thus extend their useful lifespans.

Biochemical Engineering Journal, 4, (2000), 137–141, Ganesh K et al,teaches that during the production or downstream processing of an enzymeit is always subjected to shear stress, which may deactivate the enzyme.This susceptibility of enzymes to shear stress is a major concern as itleads to the loss of enzyme activity and is, therefore, a majorconsideration in the design of the processes involving enzyme productionand its application. In this reference, cellulase enzyme was subjectedto shear stress in a stirred reactor with an objective of investigatingits deactivation under various conditions, such as different agitationspeeds, concentrations of enzyme, concentrations of buffer, pH ranges,buffer systems and the presence of gas-liquid interface. It was foundthat the extent of deactivation depends upon the conditions under whichthe enzyme was subjected to shear.

For industrial use, it is generally not sufficient for only one of theseobstacles to be overcome. For example, a stable, immobilized enzyme thatcannot be easily recovered or reused offers few advantages. Similarly,an immobilized enzyme that is easily recovered and reused, but does notmaintain its activity over an extended period offers few advantages overa process mediated by soluble enzymes, since, in both cases, the enzymesmust be replenished at frequent intervals. The main goals are to producea stable immobilized enzyme, which can also be efficiently andcompletely recovered, so that its useful lifespan is many times greaterthan the single use afforded by a soluble enzyme. An enzyme recoverysystem is therefore of paramount importance.

To date, immobilized enzyme/reactor technologies have focused on “insitu” enzyme preparations, in which the enzymes are retained within thereactors, while the process fluid is passed through. This prevents lossof the enzyme with the process fluid, and allows the enzyme to be usedseveral times. Examples include, enzymes immobilized within gels whichare used within a packed bed reactor, and enzymes attached to, orretained within, semi-permeable, hollow, fiber membranes. Enzymes havealso been incorporated within monoliths, such as those used in anautomobile catalytic converter, wherein the fluid containing thesubstrate passes through the monolith.

Unfortunately, enzymes “entrapped” within gels are subject to masstransfer limitations, which may dramatically reduce the performance ofthe immobilized enzymes, relative to soluble enzymes. The use of enzymesin packed beds is seemingly attractive in that the immobilized enzyme isretained within the reactor, while the process fluid containingsubstrate and product passes through. However, such an arrangement maybe limited by mass transfer outside the particle, due to restriction offluid flow around the tightly packed particles. To avoid extra-particlemass transfer limitations, relatively large particles are used for theimmobilized enzyme. However, larger particles lead to greater masstransfer limitations within the particle, and consequently, it isnecessary to choose a particle size that balances intraparticle andextraparticle mass transport. Furthermore, a packed-bed arrangement isonly practical if the substrate is easily transported through the packedbed. Replacing the immobilized enzyme in such a reactor may also betime-consuming as to lead to significant process “down-time”. Thus, theeconomic benefits are only realized if the enzyme is very stable, and itdoes not need frequent replacement.

Packed bed reactors and other types of “in situ” immobilized enzymereactors, such as monoliths and hollow fibers, can be prone to plugging.Consequently, they may be inappropriate if the substrate is insoluble,for e.g., in a slurry. In all cases, in situ preparations rely on thetransport of the substrate to the immobilized enzyme, either byconvection or diffusion. If flow is “segregated”, as in a slurry, orthere is insufficient mixing, or if the substrates are bulky and havelow diffusion rates, such substrate-enzyme contact may be hindered andlead to dramatically reduced efficiency and performance. Since some ofthe key industrial enzymatic processes involve slurries, e.g., starchhydrolysis and pulp processing, there is a need for an immobilizedenzyme reactor process that differs from the traditional “in situ”immobilized enzyme reactor.

A particular example of an immobilized enzyme reactor is that which isused for isomerization of dextrose to fructose. A solution of solubledextrose passes through a bed of immobilized glucose isomerase at such arate as to ensure a specific product, namely, fructose, concentration atthe bed outlet. Owing to continuous inactivation of the immobilizedenzyme, the flow rate through the packed bed reactor must becontinuously reduced, to ensure that the fructose concentration of theeffluent is held constant. It is often necessary to reduce the feed flowrate by a factor of ten or more throughout the “useful” lifespan of theimmobilized enzyme. However, such a reduction in flow rate also leads toa proportional reduction in the rate of production of fructose.Consequently, an array of generally 20 or more reactors is used, each ofwhich reactor contains immobilized glucose isomerase of a different age,and each with a different, continuously changing flow rate (H. S. Olsen,Enzymatic Production of Glucose Syrups, in Handbook of Starch HydrolysisProducts and Their Derivatives, M. W. Kearsley and S. Z. Dziedzic, eds.,Blackie Academic and Prof. Publishers (Chapman and Hall), Glasgow,1995). By combining the effluent from each reactor, the averageproduction rate of fructose is kept relatively constant. Once theimmobilized enzyme reaches the end of its useful lifespan, that reactorin the array is taken out of service, and the immobilized enzyme isreplaced with fresh enzyme.

Unfortunately, such a process arrangement is cumbersome and complex, andthe capital cost is high due to the number of reactors required, and theneed for complex valving and process control equipment to manage theadjustment of fluid flow rates to each reactor in the array. A furtherdisadvantage of such an arrangement is the production of “color” andother “off-flavor” reversion byproducts, which are more likely to begenerated at low flow rates and thus, high residence times. Thus,improvements in immobilized enzyme technology and enzyme recoverymethods could dramatically simplify this process, and improve itseconomics.

U.S. Pat. No. 5,177,005, issued Jan. 5, 1993—Lloyd and Antrim, describesa continuous process for production of fructose involving glucoseisomerase adsorbed onto a support such as a resin. The reactor waspacked with excess resin as a support and, periodically, fresh, solubleglucose isomerase was added to compensate for the inevitable loss ofactivity of the previously immobilized enzyme. In trials, fresh glucoseisomerase was added approximately every 3 weeks, on average, to keep thedextrose conversion between 40 and 44%. Over a 27 week trial, thequantity of soluble enzyme added represented approximately 4 times thequantity of enzyme originally present in the reactor. Unfortunately,such additions can only continue as long as there is binding capacity onthe resin. Once the resin is fully loaded, additional soluble glucoseisomerase may confer little benefit, unless the inactivated enzyme issomehow removed from the support. At this point, the process must eitherbe shut down to replace the gel, or run in a “variable flow rate mode”,similar to that with traditional immobilized glucose isomerase,described hereinbefore.

U.S. Pat. No. 4,033,820, issued Jul. 5, 1977—Brouillard R. E. describesanother in situ immobilized enzyme preparation based on a highly porous,spongy starch gel, modified to act as a support for the enzymes. It wasacknowledged that this approach is only suitable for substrates that aresoluble in water and that slurries cannot be processed. Anotherchallenge with this approach is the degradation of the starch supportgel. Several exotic cross-linking treatments are required if the supportis to be used for enzymes that degrade starch, e.g., amylase andglucoamylase. Bactericides, such as formaldehyde or chlorine, were usedto regularly wash the column to prevent bacterial contamination.

U.S. Pat. No. 4,209,591, issued Jun. 24, 1980 to Hendriks P., describesa multistage fluidized bed process involving countercurrent flow of animmobilized enzyme and substrate solution. The system is designed suchthat nearly all of the activity of the immobilized enzyme has been lostby the time it reaches the outlet of the multistage unit. Thus, thisoperation also involves a “single use” of enzyme, since the enzyme isnot recovered or reused. Sieve plates or mesh screens may be usedbetween and within stages to regulate the transfer of immobilized enzymeand substrate from one compartment to the next. In principal, thisprocess is amenable to the use of substrate slurries, however, theparticle sizes of the substrate and immobilized enzyme must be smallenough to pass through the sieve or mesh. Furthermore, an extremelynarrow particle size distribution is required for the immobilizedenzyme, to prevent its separation into various sized fractions duringoperation. It is well accepted that for proper fluidization, the flowrate of the substrate solution is determined in large part by the sizeand shape of particles and the particle density relative to that of thefluid.

Other examples aimed at multiple use of immobilized enzymes include U.S.Pat. No. 4,442,216, issued Apr. 10, 1984 to Harvey, D. G., U.S. Pat. No.4,511,654, issued Apr. 16, 1985 to Rohrbach et al, and U.S. Pat. No.4,594,322, issued Jun. 10, 1986 to Thompson G. J. The former describes ascrew-type reactor that includes a screw-lift mechanism and conveyor torecover the immobilized enzyme, wash it and return it to the inlet ofthe reactor. U.S. Pat. No. 4,511,654 describes an immobilizedglucoamylase packed bed reactor with subsequent substrate recycle via anultrafiltration membrane. The enzyme in this process is, thus, in situ.This process is suitable for soluble sugar feedstocks only. AforesaidU.S. Pat. No. 4,594,322 describes a similar process in which thehydrolysis products are separated into a glucose-rich stream and apolysaccharide-rich stream, the latter of which is recycled to passagain through the reactor containing immobilized enzyme.

U.S. Pat. No. 4,844,809, issued Jul. 4, 1989 to Yoshiro et al, describesthe use of a hollow fiber membrane for removal of fine particles from areaction solution. While the inventors cite this invention for otherpurposes, such as the removal of impurities, such a technique couldalso, conceivably, be used to retain enzymes immobilized to fineparticles within a hollow fiber reactor, e.g. “UltrafiltrationSeparation of Cellulase and Glucose for a Lignocellulosic Biomass toEthanol Process”, J. S. Knutsen and R. H. Davis, Conference Proceedings,Symposium on Biotechnology for Fuels and Chemicals, Breckenridge, CO,May 6–9, 2001.

Consequently, applications of immobilized enzymes have been essentiallyrestricted to in situ preparations, usually within packed bed reactors.Such processes, although technically and, occasionally economicallyfeasible, may be unnecessarily cumbersome. They may also be unsuitablefor process streams in which the substrate is present as a slurry. Animmobilized enzyme process capable of processing slurries, and/or whichavoids the complexity required to account for enzyme inactivation withinin situ preparations, e.g. production of HFCS, would be extremelyadvantageous.

Slurries cannot be processed using an in situ immobilized enzyme withina packed bed or monolith reactor, due to plugging of the bed ormonolith, and mass transfer problems that limit immobilizedenzyme-substrate contact. A design in which the immobilized enzyme isfree to circulate within the reactor can overcome these limitations.However, a means to facilitate recovery and reuse of the immobilizedenzyme is also required.

There is, therefore, a need for an immobilized enzyme recovery processwhich enables the immobilized enzyme to be reused and, preferably,recycled within a full enzymatic treatment plant, wherein substratefeedstock is enzymatically treated with particulate immobilized enzyme.

SUMMARY OF THE INVENTION

It is the object of the present invention to provide an efficientimmobilized enzyme recovery process of use in industrial plants for theenzymatic reaction of substrate materials.

Accordingly, the invention provides in one aspect an improved processfor the production of a product by the enzymatic treatment of asubstrate with a particulate, immobilized enzyme, comprising treating aprocess liquor comprising said substrate in a bioreactor to produce aslurry comprising effluent immobilized enzyme and said product in aneffluent liquor, the improvement comprising subjecting said slurry to anon-immobilized enzyme damaging shear inducing effective separationprocess to provide

-   -   a) said effluent immobilized enzyme; and    -   b) effluent liquor containing said product; and reusing said        effluent immobilized enzyme in a subsequent said enzymatic        treatment.

Surprisingly, I have discovered that notwithstanding the teachings ofthe prior art, immobilized enzymes can be separated out of bioreactorprocess effluent slurries under “shear-inducing” separation conditionshithertobefore considered to be too violent to provide undamaged enzyme.Thus, the invention provides a practical separation technique. Such a“shear-inducing” separation step includes, for example, hydrocycloning,continuous centrifuging and the like.

In a further aspect, the invention provides such separations when theeffluent slurry further comprises one or more other particulate solids.

In yet a further aspect, the invention provides an improved process forthe production of a product by the enzymatic treatment of a particulatefeed substrate with a particulate immobilized enzyme, said processcomprising treating a process liquor comprising a feed slurry of saidparticulate substrate and said particulate immobilized enzyme in abioreactor to produce said product in an effluent slurry comprisingeffluent particulate immobilized enzyme and effluent particulatesubstrate the improvement wherein said particulate substrate has aparticle size distribution different from said particulate immobilizedenzyme, and comprising

-   -   (i) subjecting said effluent slurry to a process selected from        the group consisting of screening, hydrocycloning and        centrifuging to separate said effluent particulate substrate        from said effluent slurry to provide (a) said effluent        particulate substrate, and (b) a refined slurry comprising said        effluent particulate immobilized enzyme in said process liquor;    -   (ii) subjecting said refined slurry to a process selected from        the group consisting of screening, hydrocycloning and        centrifuging to provide (c) said effluent particulate        immobilized enzyme and (d) an effluent process liquor; and    -   (iii) using said effluent immobilized enzyme in a subsequent        said enzymatic treatment.

The process of the invention provides for the recovery and reuse ofimmobilized enzymes from a process stream, including process streams inwhich there is a high solids content. Such a process entails separationof the particulate, immobilized enzyme from the process liquid, and mayalso entail separation of the solid immobilized enzyme from other solidse.g., feed material substrate within the process stream. Such a processfacilitates the use of immobilized enzymes for processes in which thesubstrate is present in a slurry, and also avoids the complexity ofprocesses currently based on in-situ enzyme preparations. The process ofthis invention leads to efficient recovery and reuse of immobilizedenzymes, and thus, allows immobilized enzymes to be used in processesthat currently can only use soluble enzymes. This dramatically reducesthe cost of enzymatic processing compared to existing soluble andimmobilized enzyme processes.

The process according to the invention as hereinabove defined applies toany enzyme immobilized to a particulate support or matrix. Ideally, thesupport used is a fine powder, with a relatively narrow particle sizedistribution. However, the use of supports with a broad particle sizedistribution is also feasible. Since the process stream may containother particulate solids, the particle characteristics of theimmobilized enzyme, e.g., size, density, tendency to aggregate, and thelike, must be sufficiently distinct from the particle characteristics ofthe process stream solids so as to permit their separation.

Preferably, the non-damaging but effective shear-inducing process of usein the practise of the invention is selected from the group consistingof hydrocycloning, continuous centrifuging and combinations thereof.

It will be readily understood to the person skilled in the art thatalthough shear-mediated deactivation of an enzyme in solution and or animmobilized enzyme slurry depends on several coupled parameters, suchas, for example, pressure drop, flow rates, residence times, rpm,G-force, vorticity and apparatus size, the skilled person can readilydetermine acceptable and efficacious process operating conditions forhis apparatus in the practise of the invention in order to significantlyreduce or eliminate the likelihood of shear-mediated deactivation of theimmobilized enzyme. Guidance is also provided for the practise of thepresent invention in aforesaid Biochemical Engineering Journal, 2000,137–141, which describes deactivation of the enzyme cellulase in asolution in a stirred reactor. The authors considered mechanical shear(stirring) and interfacial shear (gas-liquid interface), and referred toother cases of shear-mediated deactivation of cellulase. Shear wasexpressed in terms of a stirring rate, which is typical, rather than anactual shear measurement which would be in s^−1, or possibly expressedin units of surface tension as an indicator of interfacial shear.

Surprisingly, I have further discovered that unexpected efficaciousenzymatic treatment of a particulate feed substrate with a particulateimmobilized enzyme can be performed, notwithstanding the need for twoparticulate entities to collide in order to react. Accordingly, in afurther broad aspect, the invention provides an improved process for theproduction of a product by the enzymatic treatment of a particulate feedsubstrate with a particulate, immobilized enzyme, said processcomprising treating a process liquor comprising a feed slurry of saidsubstrate and said immobilized enzyme in a bioreactor to produce saidproduct in an effluent slurry comprising effluent immobilized enzyme andeffluent substrate the improvement comprising

-   -   (i) subjecting said effluent slurry to a process to separate        said effluent substrate from said effluent immobilized enzyme;    -   (ii) collecting said effluent immobilized enzyme; and    -   (iii) reusing said immobilized enzyme in said enzymatic        treatment.

In yet a further aspect, the invention provides an improved process forthe production of a product by the enzymatic treatment of a particulatefeed substrate with a particulate immobilized enzyme, said processcomprising treating a process liquor comprising a feed slurry of saidsubstrate and said immobilized enzyme in a bioreactor to produce saidproduct in an effluent slurry comprising effluent immobilized enzyme andeffluent substrate the improvement wherein said substrate has a particlesize distribution different from said immobilized enzyme, and comprising

-   -   (i) subjecting said effluent slurry to a process selected from        the group consisting of screening, hydrocycloning and        centrifuging to separate said effluent substrate from said        effluent slurry to provide (a) said effluent substrate, and (b)        a refined slurry comprising said effluent immobilized enzyme in        said process liquor;    -   (ii) subjecting said refined slurry to a process selected from        the group consisting of screening, hydrocycloning and        centrifuging to provide (c) said effluent immobilized enzyme        and (d) an effluent process liquor;    -   (iii) reusing said effluent immobilized enzyme in said enzymatic        treatment.

The aforesaid process, as hereinabove defined, comprises subjecting theslurry to non-immobilized enzyme damaging shear-inducing effectiveseparation steps.

The process of separation and recovery of the immobilized enzyme fromthe bioreactor process effluent stream may entail one or more steps,depending upon the nature of the process stream. A first step generallyinvolves the separation of the different types of solids within thebioreactor process feed stream, herein named slurry No. 1. Typically,this separation is based on particle size, but is also influenced byother particle characteristics such as, for example, density. Thisseparation produces two solid slurries, one of which contains theimmobilized enzyme. The slurry containing the effluent immobilizedenzyme, herein named slurry No. 2, is then sent for further processing,in this case, to separate the immobilized enzyme from the process fluidcontaining product. The immobilized enzyme so separated can then berecycled to the bioreactor, wherein it can be reused for furtherprocessing. The slurry containing the other process effluent solidsslurry No. 3 can be sent for further processing, or may be subjected toa second solids separation step, in the event that some of the effluentimmobilized enzyme from slurry No. 1 has been retained within thisstream. The desirability for such supplementary processing steps isinfluenced by the relative solids loading in slurry No. 1, the densitiesof the solids, and the particle size distribution of the differentsolids constituents. If subsequent solids separation is required, theeffluent immobilized enzyme recovered from this step may be added to theeffluent immobilized enzyme recovered from slurry No. 2, and returned tothe enzyme bioreactor.

The practise of the present invention is applicable, for example, to theprocesses listed in the table below.

Enzyme Substrate Product/process Amylase starch maltodextrins (corn,wheat, barley, rice..) Amylase starch modified starch for pulp sizingAmylase starch deinking of coated papers Cellulase wood pulp/re-surface-modified pulp/deinked paper cycled paper Cellulase cottonsdetergents Glucoamylase dextrins maltose/glucose Glucose glucosefructose (high fructose corn syrup) isomerase Glucose glucose gluconicacid oxidase Protease proteins detergents Tyrosinase L-tyrosine L-DOPAXylanase wood pulp/xylan biobleaching of wood pulp Xylanase xylan Xylose

Examples of processes involving solid substrates and immobilized enzymesinclude:

-   (1) amylases used to partially or completely hydrolyze starch to    produce either modified starch or dextrins, the latter of which may    be subject to further processing to produce simple sugars, (2)    cellulases and/or xylanases used to partially or completely    hydrolyze pulp fibers, as in enzymatic deinking, biobleaching, fiber    modification, or glucose/xylose production, for subsequent    fermentation, and-   (3) cellulases, lipases, or proteases used in detergents for fiber    modification and/or stain removal.

Examples of processes involving soluble substrates and immobilizedenzymes include glucose oxidase used to convert glucose to gluconicacid, glucoamylase used to convert dextrins to monosacharrides, glucoseisomerase used to produce high fructose corn syrups from glucose,tyrosinase used to produce L-DOPA from L-tyrosine or to convert othermono- or di-phenolics in wastewater streams, and enzymes such as lipasesused to produce nutraceuticals.

BRIEF DESCRIPTION OF THE DRAWINGS

In order that the invention may be better understood, preferredembodiments will now be described, by way of example only, withreference to the accompanying drawings, wherein

FIGS. 1–5 represent process flow charts of various embodiments ofprocesses according to the invention;

FIG. 6 represents a graph of hydrocyclone assays with spezyme® solubleenzyme;

FIG. 7 represents a graph of hydrocyclone assays with liquozyme® solubleenzyme; and when the same letters denote like parts and process steps.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS EXAMPLE 1

Reference is now made to FIG. 1 wherein the process steps are as listedhereinbelow.

-   A=Process stream (slurry) containing immobilized enzyme and other    process solids-   B=Overflow, primarily large solids with very little process liquid-   C=Underflow, comprised of fine solids and most of the process liquid    from stream A-   D=overflow, comprised almost entirely of process liquid-   E=underflow, comprised of fine solids and a small portion of process    liquid.

The flow diagram of FIG. 1 is based on the premise that the immobilizedenzyme is present as a fine solid, while the other process solids arepresent as much larger particles. This implies that stream C containsthe immobilized enzyme to ultimately be recycled to the reactor for usein a subsequent enzymatic reaction. Should the opposite be true, streamB would contain the immobilized enzyme, which would then be recycled,and the solid separation of stream C provided via the hydrocyclone maynot be necessary.

A mixture of 30% corn mash (mean 700 μm; 98%>350 μm) and 1.33%immobilized enzyme (mean 18 μm; range 1 to 120 μm) was processedaccording to FIG. 1. A 355 μm mesh screen was used to separate the twosolids. 95% of the corn mash and 7% of the immobilized enzyme proceededto the overflow (stream B). The balance (as stream C) proceeded to amanifolded set of 10 mm hydrocyclones with a 2.5 mm (dia) inlet. 65% ofthe solids in stream C were directed to the underflow (stream E), and35% proceeded to stream D, based on a pressure drop of 2.7 bar acrosseach hydrocyclone, and a flow rate of 2.8 L/min/cyclone. 78% of theprocess fluid in stream C was directed to stream D. The total recoveryof immobilized enzyme under these conditions was, thus, 60%.

EXAMPLE 2

With reference to FIG. 1 wherein the process steps are as listedhereinbelow.

-   A=Process stream (slurry) containing immobilized enzyme and other    process solids-   B=Overflow, primarily large solids with very little process liquid-   C=Underflow, comprised of fine solids and most of the process liquid    from stream A-   D=overflow, comprised almost entirely of process liquid-   E=underflow, comprised of fine solids and a small portion of process    liquid.

This flow diagram is based on the premise that the immobilized enzyme ispresent as a fine solid, while the other process solids are present asmuch larger particles. This implies that stream C contains theimmobilized enzyme to ultimately be recycled to the reactor. Should theopposite be true, stream B would contain the immobilized enzyme, whichwould then be recycled, and the solid separation of stream C providedvia the hydrocyclone may not be necessary.

A mixture of 30% corn mash (mean 700 μm; 98%>350 μm) and 2.67%immobilized enzyme (mean 18 μm; range 1 to 120 μm) was processedaccording to FIG. 1. A 355 μm mesh screen was used to separate the twosolids. 90% of the corn mash and 6% of the immobilized enzyme proceededto the overflow (stream B). The balance (as stream C) proceeded to amanifolded set of 10 mm hydrocyclones with a 2.5 mm (dia) inlet. 81% ofthe solids in stream C were directed to the underflow (stream E), and19% proceeded to stream D, based on a pressure drop of 2.7 bar acrosseach hydrocyclone, and a flow rate of 2.8 L/min/cyclone. 73% of theprocess fluid was directed to stream D. The total recovery ofimmobilized enzyme under these conditions was 76%.

EXAMPLE 3

With reference to FIG. 1 wherein the process steps are as listedhereinbelow.

-   A=Process stream (slurry) containing immobilized enzyme and other    process solids-   B=Overflow, primarily large solids with very little process liquid-   C=Underflow, comprised of fine solids and most of the process liquid    from stream A-   D=overflow, comprised almost entirely of process liquid-   E=underflow, comprised of fine solids and a small portion of process    liquid.

This flow diagram is based on the premise that the immobilized enzyme ispresent as a fine solid, while the other process solids are present asmuch larger particles. This implies that stream C contains theimmobilized enzyme to ultimately be recycled to the reactor. Should theopposite be true, stream B would contain the immobilized enzyme, whichwould then be recycled, and the solid separation of stream C providedvia the hydrocyclone may not be necessary.

A mixture of 30% corn mash (mean 700 μm; 98%>350 μm) and 2.0%immobilized enzyme (mean 18 μm; range 1 to 120 μm) was processedaccording to FIG. 1. A 250 μm mesh screen was used to separate the twosolids. 100% of the corn mash and 4% of the immobilized enzyme proceededto the overflow (stream B). The balance (as stream C) proceeded to amanifolded bank of 10 mm hydrocyclones with a 2.5 mm (dia) inlet. 71% ofthe solids in stream C were directed to the underflow (stream E), and29% proceeded to stream D, based on a pressure drop of 2.7 bar acrosseach hydrocyclone, and a flow rate of 2.8 L/min/cyclone. 82% of thefluid in stream C was directed to stream D, and 18% of the fluid went tostream E. The total immobilized enzyme recovery under these conditionswas 68%.

EXAMPLE 4

With reference to FIG. 4 wherein the process steps are as listedhereinbelow.

-   A=Process stream (slurry) containing immobilized enzyme and other    process solids-   B=overflow, primarily large solids with very little process liquid-   C=underflow, comprised of fine solids and most of the process liquid    from stream A-   D=overflow, comprised almost entirely of process liquid-   E=underflow, comprised of fine solids and a small fraction of the    liquid from stream C.-   F=process liquid for recycle

This flow diagram is based on the premise that the immobilized enzyme ispresent as a fine solid, while the other process solids are present asmuch larger particles. This implies that stream C contains theimmobilized enzyme to ultimately be recycled to the reactor. Should theopposite be true, stream B would contain the immobilized enzyme, whichwould then be recycled. The solid separation of stream C provided viathe hydrocyclone may be required to dilute the solids present in streamA, facilitating the separation of fine and coarse solids via the screen.

A mixture of 30% corn mash (mean 700 μm; 98%>350 μm) and 2.0%immobilized enzyme (mean 25 μm; range 5 to 90 μm) was processedaccording to FIG. 4. After dilution, the feed to the screen contained15% corn mash and 1% immobilized enzyme. A 250 μm mesh screen was usedto separate the two solids. 100% of the corn mash and 1% of theimmobilized enzyme proceeded to the overflow (stream B). The balance (asstream C) proceeded to a manifolded bank of 10 mm hydrocyclones with a2.5 mm (dia) inlet. 78% of the solids in stream C were directed to theunderflow (stream E), and 22% proceeded to stream D, based on a pressuredrop of 2.7 bar across each hydrocyclone, and a flow rate of 2.8L/min/cyclone. 72% of the fluid in stream C was directed to stream D,and 28% of the fluid went to stream E. The total immobilized enzymerecovery under these conditions was 77%.

EXAMPLE 5

With reference to FIG. 2 wherein the process steps are as listedhereinbelow.

-   A=Process stream (slurry) containing immobilized enzyme and other    process solids-   B=underflow, primarily large solids with some process liquid-   C=overflow, comprised of fine solids and 50–80% of the process    liquid from stream A-   D=overflow, comprised almost entirely of process liquid-   E=underflow, comprised of fine solids and a small fraction of the    liquid from stream C.

This flow diagram is based on the premise that the immobilized enzyme ispresent as a fine solid, while the other process solids are present asmuch larger particles. This implies that stream C contains theimmobilized enzyme to ultimately be recycled to the reactor. Should theopposite be true, stream B would contain the immobilized enzyme, whichwould then be recycled, and the solid separation of stream C providedvia the hydrocyclone may not be necessary.

A mixture of 30% corn mash (mean 700 μm; 98%>350 μm) and 1% immobilizedenzyme (mean 18 μm; range 1 to 120 μm) was processed according to FIG.2. A hydrocyclone with a 3 cm (dia) inlet was used to separate the twosolids. The feed rate was 6.6 L/s, and the pressure drop across thehydrocyclone was 0.5 bar. 100% of the corn mash and 29% of theimmobilized enzyme proceeded to the underflow (stream B). The balance(as stream C) proceeded to a manifolded set of 10 mm hydrocyclones witha 2.5 mm (dia) inlet. 78% of the solids in stream C were directed to theunderflow (stream E), and 22% proceeded to stream D, based on a pressuredrop of 3.4 bar across each hydrocyclone, and a flow rate of 9.4L/min/cyclone. The total recovery of immobilized enzyme under theseconditions was thus 55%.

EXAMPLE 6

With reference to FIG. 3 wherein the process steps are as listedhereinbelow.

-   A=Process stream (slurry) containing immobilized enzyme and other    process solids-   B=underflow, primarily large solids with some process liquid-   C=overflow, comprised of fine solids and 50–80% of the process    liquid from stream A-   D=overflow, comprised almost entirely of process liquid-   E=underflow, comprised of fine solids and a small fraction of the    liquid from stream C.-   F=process liquid for recycle

This flow diagram is based on the premise that the immobilized enzyme ispresent as a fine solid, while the other process solids are present asmuch larger particles. This implies that stream C contains theimmobilized enzyme to ultimately be recycled to the reactor. Should theopposite be true, stream B would contain the immobilized enzyme, whichwould then be recycled, and the solid separation of stream C providedvia the hydrocyclone may not be necessary.

A mixture of 30% corn mash (mean 700 μm; 98%>350 μm) and 1.33%immobilized enzyme (mean 18 μm; range 1 to 120 μm) was processedaccording to FIG. 3, with sufficient fluid recycle (stream F) to reducethe solids loading to the first hydrocyclone to ˜20%. A hydrocyclonewith a 3 cm (dia) inlet was used to separate the two solids. The feedrate was 10.1 L/s, and the pressure drop across the hydrocyclone was 0.5bar. 100% of the corn mash and 18% of the immobilized enzyme proceededto the underflow (stream B). The balance (as stream C) proceeded to amanifolded set of 10 mm hydrocyclones with a 2.5 mm (dia) inlet. 78% ofthe solids in stream C were directed to the underflow (stream E), and22% proceeded to stream D, based on a pressure drop of 2.7 bar acrosseach hydrocyclone, and a flow rate of 2.8 L/min/cyclone. The totalrecovery of immobilized enzyme under these conditions was, thus, 64%.

EXAMPLE 7

With reference to FIG. 5 wherein the process steps are as listedhereinbelow.

-   A=Process stream (slurry) containing immobilized enzyme and other    process solids-   B=underflow, primarily large solids with some process liquid-   C=overflow, comprised of fine solids and 50–80% of the process    liquid from stream A-   D=overflow, comprised almost entirely of process liquid-   E=underflow, comprised of fine solids and a small fraction of the    liquid from stream D.-   F=process liquid for recycle-   D′=overflow, comprised almost entirely of process liquid-   E′=underflow, comprised of fine solids and a small fraction of the    liquid from stream C.-   F′=process liquid for recycle

This flow diagram is based on the premise that the immobilized enzyme ispresent as a fine solid, while the other process solids are present asmuch larger particles. This implies that stream C contains theimmobilized enzyme to ultimately be recycled to the reactor. Should theopposite be true, stream B would contain the immobilized enzyme, whichwould then be recycled, although the solid separation of streams C and Dvia the hydrocyclones may be needed to produce recycle fluid to dilutethe solids in stream A.

Note that FIG. 5 differs from FIG. 3 only by the fact that an additionalhydrocyclone is added to improve the separation/recovery of fineparticles, and to increase the percentage of process fluid recycled tomix with stream A. As required, additional hydrocyclones/centrifugesbeyond the two shown here can be incorporated into the process, addingto the fluid recycle streams F and F′, and solid recycle streams E andE′.

A mixture of 30% corn mash (mean 700 μm; 98%>350 μm) and 1.33%immobilized enzyme (mean 18 μm; range 1 to 120 μm) was processedaccording to FIG. 5, with sufficient fluid recycle (streams F and F′) toreduce the solids loading to the first hydrocyclone to ˜10%. Ahydrocyclone with a 4.8 cm (dia) inlet was used to separate the twosolids. The feed rate was 21 L/s, and the pressure drop across thehydrocyclone was 0.5 bar. 100% of the corn mash and 8% of theimmobilized enzyme proceeded to the underflow (stream B). The balance(as stream C) proceeded to a manifolded set of 10 mm hydrocyclones witha 2.5 mm (dia) inlet. 78% of the solids in stream C were directed to theunderflow (stream E), and 22% proceeded to stream D, based on a pressuredrop of 2.7 bar across each hydrocyclone, and a flow rate of 2.8L/min/cyclone. A second set of hydrocyclones was used to separate thesolids in stream D, directing 70% of the solids to the underflow (streamE′), and 30% to the overflow (stream F′). For each of these latter twohydrocyclones, 73% of the fluid was directed to the overflow (streams Fand F′), and 23% was directed to the underflow (E and E′). The totalrecovery of immobilized enzyme under these conditions was thus 86%.

EXAMPLE 8

With reference to FIG. 1 wherein the process steps are as listedhereinbelow.

-   A=Process stream (slurry) containing immobilized enzyme and other    process solids-   B=Overflow, primarily large solids with very little process liquid-   C=Underflow, comprised of fine solids and most of the process liquid    from stream A-   D=overflow, comprised almost entirely of process liquid-   E=underflow, comprised of fine solids and a small portion of process    liquid.

This flow diagram is based on the premise that the immobilized enzyme ispresent as a fine solid, while the other process solids are present asmuch larger particles. This implies that stream C contains theimmobilized enzyme to ultimately be recycled to the reactor. Should theopposite be true, stream B would contain the immobilized enzyme, whichwould then be recycled, and the solid separation of stream C providedvia the hydrocyclone may not be necessary.

A mixture of 30% corn mash (mean 700 μm; 98%>350 μm) and 2.66%immobilized enzyme (mean 140 μm; range 1 to 220 μm; median 120 μm) wasprocessed according to FIG. 1. A 355 μm mesh screen was used to separatethe two solids. 99% of the corn mash and 1% of the immobilized enzymeproceeded to the overflow (stream B). The balance (as stream C)proceeded to a manifolded set of 10 mm hydrocyclones with a 2.5 mm (dia)inlet. 96% of the solids in stream C were directed to the underflow(stream E), and 4% proceeded to stream D, based on a pressure drop of2.7 bar across each hydrocyclone, and a flow rate of 2.8 L/min/cyclone.71% of the process fluid in stream C was directed to stream D. The totalrecovery of immobilized enzyme under these conditions was, thus, 95%.

EXAMPLE 9

Diluted Feed

A mixture of 17% corn mash (mean 750 μm; 85%>350 μm) and 1.33%immobilized enzyme (mean 120 μm; range 75 to 200 μm) was processedaccording to FIG. 1, at a feed rate of 225 L/min. A screen with d50=186μm was used to separate the solids. Ninety percent of the solids <355 μmproceeded to the underflow (stream C), with 6% of the immobilized enzymelost to the overflow (stream B). Stream C was processed as a batchthrough a manifolded set of fifteen 10 mm hydrocyclones, with a pressuredrop of 5.4 bar across each hydrocyclone, and a flowrate of 181 L/min.Seventy percent of the process fluid was directed to stream D, and 54%of the solids in stream C were directed to stream E. The total recoveryof immobilized enzyme under these conditions was 50%.

EXAMPLE 10

Double Deck Screen

A stream containing 35% solids (96% corn mash) was processed accordingto FIG. 1, with double decked screens. The top screen has a d50=562 μm,and the bottom screen has a d50 of 294 μm. The solids are thus subjectto a “rough” cut on the top screen, and a “fine” cut on the bottomscreen. Eighty nine percent of the solids <355 μm proceeded to stream C,and 12% of the immobilized enzyme was lost to stream B. Stream C wassubsequently processed at 132 L/min, with a pressure drop of 5.6 bar;87% of the fluid was directed to overflow stream D. The total recoveryof immobilized enzyme was 69%.

EXAMPLE 11

Two Stage Screen

A stream containing 35% solids (96% corn mash) was processed accordingto FIG. 1, modified to use two screens arranged in series on shakersinclined at 30 degrees with respect to horizontal. The first screen hada d50 of 863 μm, and the second screen had a d50 of 387 μm. The solidsare thus subject to a “rough” cut on the first screen, and a “fine” cuton the second screen. The feed rate was 132 L/min to the first screen;the screen unders were allowed to accumulate and then fed to the secondscreen at a rate of 107 L/min. Eighty seven percent of the solids <355μm and ninety four percent of the immobilized enzyme were ultimatelydirected to stream C for separation via the hydrocyclones, as describedin Example 2, hereinabove. The total recovery of immobilized enzyme was90%.

EXAMPLE 12

Soluble Substrate Stream

An immobilized enzyme was recovered from a soluble substrate streamaccording to essentially FIG. 1, without the screen. The loading ofimmobilized enzyme was 2.1 g/L. The suspension was fed to a manifoldedset of fifteen 10 mm hydrocyclones, at a rate of 147 L/min, with apressure drop of 3.8 bar. Approximately four percent of the fluid wasdirected to the hydrocyclone underflow, wherein the solids concentrationwas 56 g/L. The recovery of immobilized enzyme was thus 97%.

EXAMPLE 13

Recycle Operation

A stream containing 30% solids (97% corn mash) was processed accordingto FIG. 4. The screen had a d50 of 387 μm. Slurry was fed at a rate of93 L/min, leading to a screen unders flowrate stream C of 140 L/min witha stream F recycle rate of 76 L/min. Eight percent of the solids <355 mmwere lost to stream B, which included 5% of the immobilized enzyme.Eighty eight percent of the <250 μm solids in stream C were recovered instream E.

EXAMPLE 14

Dynamic Drainage Jar

A stream containing 1% hardwood pulp and 0.5 g/L immobilized cellulase(mean diameter 6 μm) was mixed at 800 rpm. The suspension was thenrapidly drained through a 100 μm screen. Eighty five percent of theimmobilized enzyme was recovered in the underflow/filtrate.

EXAMPLE 15

Large Particle Immobilized Enzyme

A mixture of 22% corn mash (mean 650 μm; 93%>350 μm) and 1.6%immobilized enzyme (range 850 to 1200 μm) was processed according toFIG. 1, at a feed rate of 50 L/min. An 850 μm screen was used toseparate the solids. Virtually all of the solids >850 μm proceeded tothe overflow (stream B); only 0.02% of the solids in stream C werelarger than 850 μm. This implies that there was essentially 100%recovery of the immobilized enzyme in stream B. Stream C was sent forfurther processing, while stream B, with the immobilized enzyme, wasrecycled for further use.

EXAMPLE 16

Two examples, hereinbelow denoted 16A and 16B describe experiments toestablish effect of shear on enzymes.

Description of experiments:

Equipment and materials

The equipment consisted of a 250 L feed tank with mixer, a positivedisplacement pump (maximum 6.8 bar discharge, capacity up to 22 L/min),and a manifolded set of six hydrocyclones (10 mm, with a 2.5 mm (dia)inlet). The piping was arranged to return the fluid from the overflowand underflow of the hydrocyclone to the feed tank, so that theapparatus was run under a continuous recycle. The feed tank wasinitially filled with 150 L of soluble enzyme; a sample of this solutionwas collected for a subsequent activity assay. In the first experiment,Spezyme® enzyme from Genencor International was used, and in the secondexperiment, Liquozyme® enzyme from Novozymes was used. The activity ofeach α-amylase was determined using a reducing sugars assay, using cornflour as the substrate.

Procedure:

An aforesaid soluble enzyme was pumped through the hydrocyclones at arate of 16 L/min, with a pressure drop of 2.7 bar between the inlet andthe outlets. Seventy percent of the fluid was directed to the overflow,and thirty percent was directed to the underflow. Samples of the solubleenzyme were collected at regular intervals, and assayed for enzymeactivity, as follows:

1.0 mL of enzyme sample was added to 24.0 mL of buffer either pH 6.9 forSpezyme® enzyme, or pH 5.0 for Liquozyme® enzyme. The reaction wasinitiated by adding 0.20 g of corn flour. Samples were collected at timezero, and every 3 minutes for 15 minutes. Samples were centrifuged toprecipitate any suspended corn flour. 1.5 mL of the supernatant wasmixed with 3 mL of dinitrosalicylic acid reagent in a test tube, andcooked for 5 minutes in a boiling water bath. The mixture was thencooled to room temperature, and the absorbance was determined at 540 nm.

Results

16A

1) Experiments with Spezyme® Enzyme

The results from experiments with Spezyme® enzyme are presented in FIG.6. The data show that processing the soluble enzyme through thehydrocyclone leads to approximately a 40% reduction in activity within15 minutes, and approximately a 75% reduction in activity after 90minutes. In contrast, under normal storage conditions, the enzyme isexpected to remain active over a period of 4 to 6 months (manufacturer'stechnical sheet).

16B

2) Experiments with Liquozyme® Enzyme

The results from experiments with Liquozyme® enzyme are presented inFIG. 7, below. The data show that processing the soluble enzyme throughthe hydrocyclone leads to approximately a 25% reduction in activitywithin 15 minutes, but no further loss of activity thereafter. Incontrast, under normal storage conditions, the enzyme is expected toremain active over 4 to 6 months (manufacturer's technical sheet).

Thus, it can be seen that the shear results shown in FIGS. 6 and 7 forthe processing of these soluble enzymes through the hydrocyclone system,leads to a rapid loss of activity, although, Liquozyme® enzyme is lesssensitive to shear inactivation than Spezyme® enzyme.

Although this disclosure has described and illustrated certainembodiments of the invention, it is to be understood that the inventionsis not restricted to those particular embodiments. Rather, the inventionincludes all embodiments which are functional or mechanical equivalenceof the specific embodiments and features that have been described andillustrated.

1. A process for producing a product by enzymatic treatment of aparticulate substrate comprising: providing a particulate feedsubstrate, providing a particulate, immobilized enzyme, reacting theparticulate feed substrate with the particulate, immobilized enzyme in abioreactor to produce an effluent process liquor containing the effluentparticulate, immobilized enzyme, a product of the reaction and aneffluent particulate substrate, which has a particle size distributiondifferent from the effluent particulate, immobilized enzyme, treatingthe process liquor to produce a slurry, subjecting the slurry to atleast one of screening, continuous centrifuging or hydrocycloning toseparate the effluent particulate substrate from the effluentparticulate, immobilized enzyme, subjecting the slurry to hydrocycloningto separate the particulate, immobilized enzyme from the product, andrecycling the particulate, immobilized enzyme.
 2. The method of claim 1,wherein the particulate feed substrate is corn mash.
 3. The method ofclaim 1, wherein the particulate feed substrate is cellulosic feedstock.
 4. The method of claim 1, wherein the particulate feed substrateis hemicellulosic feed stock.